Plant beneficial traits of endophytic bacteria associated with fennel (Foeniculum vulgare Mill.)

In this study, we used 16S rRNA gene sequence analysis to describe the diversity of cultivable endophytic bacteria associated with fennel (Foeniculum vulgare Mill.) and determined their plant-beneficial traits. The bacterial isolates from the roots of fennel belonged to four phyla: Firmicutes (BRN1 and BRN3), Proteobacteria (BRN5, BRN6, and BRN7), Gammaproteobacteria (BRN2), and Actinobacteria (BRN4). The bacterial isolates from the shoot of fennel represented the phyla Proteobacteria (BSN1, BSN2, BSN3, BSN5, BSN6, BSN7, and BSN8), Firmicutes (BSN4, BRN1, and BRN3), and Actinobacteria (BRN4). The bacterial species Bacillus megaterium, Bacillus aryabhattai, and Brevibacterium frigoritolerans were found both in the roots and shoots of fennel. The bacterial isolates were found to produce siderophores, HCN, and indole-3-acetic acid (IAA), as well as hydrolytic enzymes such as chitinase, protease, glucanase, and lipase. Seven bacterial isolates showed antagonistic activity against Fusarium culmorum, Fusarium solani, and Rhizoctonia. solani. Our findings show that medicinal plants with antibacterial activity may serve as a source for the selection of microorganisms that exhibit antagonistic activity against plant fungal infections and may be considered as a viable option for the management of fungal diseases. They can also serve as an active part of biopreparation, improving plant growth.

Production of fennel through eco-friendly technology is an important approach, ensuring organic fennel.The application of plant-beneficial microbes is considered as an alternative eco-friendly approach to improving medicinal plant health [16][17][18].Among these microbes, endophytic bacteria that colonize plant internal tissues, roots, leaves, and stems can provide beneficial effects to plants [19][20][21].There are many reports on the diversity of endophytic bacteria associated with medicinal plants, and their biological activity has been reported, e.g., Ziziphora capitata, Hypericum perforatum [16], Aloe vera, [22], and Origanum vulgare [23].Endophytes colonizing plant tissue are assumed to play an important role in the synthesis of biologically active compounds and also protect plants from soilborne disease [24][25][26].Several mechanisms underlying plant beneficial effects have been reported, including the production of phytohormones, cell wall-degrading enzymes, hydrogen cyanide (HCN), and ACC deaminase [27,28].Moreover, there is evidence that the chemical composition of the exudate affects the microbial diversity and activity associated with plants [29].For example, bacteria associated with medicinal plants such as Matricaria chamomilla, Baccharoides anthelmintica, and Calendula officinalis exhibit antimicrobial activity similar to that of the host plant [30][31][32].
To date, there have been only a few reports of endophytes associated with fennel and their beneficial effects on plants, despite numerous studies reporting on the phytochemical contents and biological activity of fennel (Foeniculum vulgare Mill.).To enhance our understanding of the function of endophytes in plant growth and development, it is crucial to gain knowledge about the physiological activities of endophytic bacteria associated with medicinal plants.In the current study, we aim (1) to isolate and identify culturable endophytic bacteria associated with fennel by using 16S rRNA gene analysis, and (2) to evaluate their plant-beneficial properties.

Plant sample collection
In June 2019, fennel (Foeniculum vulgare Mill.) was harvested from Ugam-Chatkal State Biosphere Reserve, Uzbekistan (41°15'27.7"N,69°54'41.4"E),a remote and forested region situated in the Western Tien Shan province.Ten individual plants with their root systems were collected using sterile gloves at a distance of 12-15 m.They were then stored in zip-lock plastic bags and brought to the lab for additional analysis.

Isolation of endophytic bacteria
For sterilization of plant roots and leaves, 10% NaClO and 70% ethanol were used.Then, they were rinsed in 2 L of sterile water (2 min) five times.The root and leaves (10 g each) were squeezed out with a sterile mortar and mixed with 90 mL of phosphate buffer solution [33].The mixtures resulting from dilutions (10 1 -10 5 ) were spread out in 100 µL of tryptic soy agar (TSA) (BD, Difco Laboratories, USA) with an addition of 50 µg/mL of nystatin and stored in a thermostat for 96 h at 28 °C.Every single colony that had a distinct color, shape, surface, and consistency was the source of the new isolates, and the plates were examined for bacterial growth.

Identification of bacteria
The heat treatment method was used to isolate bacterial DNA [34] as follows: The bacterial isolates were cultivated on Petri plates with TSA at 28 °C for 72 h.Subsequently, the colonies were transferred into Eppendorf tubes with 300 µL of sterile Milli-Q water, incubated at 90 °C for 20 min in a dry block heater (IKA Works, Inc., Wilmington, USA), and centrifuged at 12,000 rpm for 5 min.The presence of DNA in the tubes was tested using gel electrophoresis and quantified with NanoDrop™ One (Thermo Fisher Scientific Inc., Waltham, USA).
The 16S rRNA gene sequences were checked for identity with the relative sequences from the GenBank of NCBI (http://www.ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool (BLAST).The Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) online software was used for multiple alignments of all obtained and relative 16S rRNA gene sequences.The maximum composite likelihood method [37] was used for counting the evolutionary distances.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches of a phylogenetic tree, which was built using MEGA X software [38].
Upon the deposition of the obtained 16S rRNA gene sequences to GenBank, they were assigned the following accession numbers: MT310821-MT310835.

Antifungal activity of endophytes
The ability of cell-free solutions of endophytic bacterial isolates and plant extracts to inhibit plant pathogenic fungi Rhizoctonia solani, Fusarium culmorum (Wm.G.Sm.)Sacc., and F. solani (Mart.)Sacc.J.G. Kühn was investigated in the way detailed by Egamberdieva et al. [39].
The bacterial isolates were grown in TSB broth for three days, and 50 μL of bacterial cultures were dropped into a hole in PDA plates (4 mm in diameter).Fungal strains were obtained from the culture collection of microorganisms at the National University of Uzbekistan, and they were grown in PDA plates at 28 °C for five days.Disks of fresh fungus cultures (5 mm in diameter) were cut out and placed 2 cm away from the hole filled with bacterial filtrate.The plates were sealed with Parafilm®M and incubated at 28 °C in darkness until the fungi had grown over the control plates without bacteria.Antifungal activity was recorded as the width of the growth-inhibition zone between the fungus and the test bacterium.

Plant-beneficial traits of endophytes
On TSA media, the ability of bacterial isolates to produce hydrogen cyanide (HCN) was examined.The color change of filter paper immersed in a 1% picric acid and 2% sodium carbonate solution and put on Petri plates was measured [40].The bacterial isolates' ability to produce siderophores was determined using the following method described by Schwyn and Neilands [41].Protease secretion was revealed by growing strains on TSA plates (20 times diluted) amended with skimmed milk to a final concentration of 5%.The halo appearing on the first to the second day of cultivation around colonies indicated the presence of extracellular protease [42].Furthermore, β-1,3 and β-1,4glucanase activity was tested using the substrate lichenan (Sigma-Aldrich, St. Louis, MO) in top agar plates (Walsh et al. 1995).The production of chitinase by bacterial isolates was determined on colloidal chitin medium using the Malleswari and Bagyanarayana [44] method.The lipase activity of bacterial strains was determined by the Tween lipase indicator assay.Bacterial strains were grown in LC agar (LB agar containing 10 mM MgSO4 and 5 mM CaCI2) containing 2% Tween 80 at 28 °C [45].After five days, the degradation of Tween was taken as a clear halo around the bacterial inoculum.Using the technique outlined by Bano and Musarrat [46], the synthesis of IAA (indole 3-acetic acid) by endophytic isolates was investigated.The IAA concentration in culture was calculated by using a calibration curve of pure IAA as a standard (Sigma-Aldrich, Merck).According to Egamberdieva and Kucharova's description [47], ACC deaminase synthesis was investigated with 1-aminociclopropane-1-carboxylacid (ACC) as the only N source.The P-solubilization ability of bacterial isolates was performed as previously described by Chen et al. [48].

Plant growth promotion
After being cultured for 72 h in tryptic soy broth (TSB; Sigma-Aldrich), the bacterial cultures were adjusted to an optical density of 0.1 (OD620 = 0.1) at 620 nm, which corresponds to approximately 10 8 cells/mL.The fennel seeds were dipped into bacterial solutions and, after 5 min, inoculated seeds with bacteria were sown in pots (two seeds per pot) (12 cm in diameter and 16 cm in depth) filled with 500 g of soil.After germination, one seedling was kept per pot.In the experiment, a randomized design was employed, with each treatment consisting of 10 pots.There were two treatments in the pot experiment: pots with the plant uninoculated with bacteria and pots with plants inoculated with bacteria.The plants were grown for two weeks at 24-26 °C during the day and 17-18 °C at night, with 40% humidity.The shoot and root lengths as well as the dry weight were measured [47].

Statistical analyses
Using Microsoft Excel 2010's analysis of variance software, the data were examined for statistical significance.Data obtained from the plant growth test were subjected to analysis of variance (ANOVA) with SPSS software (version 15) at p < 0.05.The results are presented as average means and standard error (SE).The difference between means was compared by a high-range statistical domain (HSD) using Tukey's test.The treatment means were separated by the least significant difference (LSD) test at p < 0.05.

Isolation and identification of cultivable endophytic bacteria
In total, 60 bacterial isolates were obtained from the plant tissues of fennel.The RFLP analysis was utilized for the selection of similar isolates.After RFLP analysis, 18 bacterial isolates were selected (7 from roots and 11 from shoots) and siblings were removed.The colonies of some isolates with plant-beneficial traits are shown in Figure 1.
All isolates were determined using the BLAST basic local alignment search tool and matched with correlative strains from the NCBI GenBank.The isolates were 98.95%-99.93%identical to their closest relatives registered in GenBank®.Sequence similarities of endophyte bacteria isolated from the root and shoot systems of fennel are given in Tables 1 and 2. The length of the identified nucleotide sequences of 16S rRNA gene in the isolates varied from 1408 to 1470 bp and was noted as adequate for confidential identification based on 16S rRNA gene analysis using the BLAST tool.All isolated strains got their accession numbers (Tables 1 and 2).As shown in Table 1, the roots of fennel harbored seven species belonging to four phyla: Firmicutes (BRN1 and BRN3), Proteobacteria (BRN5, BRN6, and BRN7), Gammaproteobacteria (BRN2), and Actinobacteria (BRN4).Table 2 comprises 11 strains isolated from shoots of fennel and represents the phyla Proteobacteria (BSN1, BSN2, BSN3, BSN5, BSN6, BSN7, and BSN8), Firmicutes (BSN4, BRN1, and BRN3) and Actinobacteria (BRN4) (Figure 2).Above all, Bacillus megaterium, Bacillus aryabhattai, and Brevibacterium frigoritolerans were found both in the roots and shoots of fennel.

Plant growth-promoting activity of endophytic bacteria
The isolated endophytes were tested for their ability to stimulate the growth of fennel seedlings (Table 4).Some of the tested bacteria showed high plant-growth promotion in fennel seedlings.Seed inoculation with strain B. aryabhattai BRN3 resulted in a 21.5% and 24.5% increase in shoot and root length, respectively as compared to the control.The shoot and root dry mass also rose to 25.2% and 24.6%, respectively, as compared to the control.The strains B. megaterium BRN1, P. reinekei BRN2, P. jessenii BRN6, P. plecoglossicida BRN7, K. pneumoniae BSN2, and S. maltophilia BSN6 were less effective, and increased shoot length up to 7.6%-17.7%and root length up to 6.1%-18.4% in comparison with control.The strains P. chlororaphis BSN8, P. lini BRN5, B. simplex BSN4, K. pasteurii BSN5, P. putida BSN7, and B. frigoritolerans BRN4 did not exhibit, or showed very low, plant growth-promoting activity.Two strains (E.mori BSN1 and E. cloacae BSN3) inhibited the growth of fennel seedlings and reduced shoot and root length and dry weight.

Plant-beneficial traits
Detailed results of plant-beneficial traits of endophytic bacteria isolated from fennel (Foeniculum vulgare Mill.) are given in Table 5.According to the results, B. megaterium BRN1, P. reinekei BRN2, B. aryabhattai BRN3, P. jessenii BRN6, P. plecoglossicida BRN7, K. pneumoniae BSN2, S. maltophilia BSN6, and P. putida BSN7 produced IAA.Among these bacterial strains, the highest IAA synthesis was demonstrated in the root-and shoot-associated bacteria B. aryabhattai BRN3.Siderophore production was observed in 8 out of 15 bacterial isolates.Seven isolates out of 15 showed ACC deaminase production and phosphate solubilization.Nine of the strains also showed hydrogen cyanide (HCN) production.The strains were also tested for fungi cell wall-degrading enzymes (chitinase, glucanase, protease, and lipase) production.It was revealed that strains S. maltophilia BSN6, P. jessenii BRN6, B. simplex BSN4, and P. reinekei BRN2 produced three out of four tested enzymes.The strains P. chlororaphis BSN8, P. plecoglossicida BRN7, P. putida BSN7, and P. lini BRN5 showed production of two enzymes.The strains E. cloacae BSN3, K. pneumoniae BSN2, and B. aryabhattai BRN3 produced only one of the tested enzymes.The strains K. pasteurii BSN5, B. frigoritolerans BRN4, E. mori BSN1, and B. megaterium BRN1 did not produce any of the tested enzymes."+" positive, "-" negative", R: bacteria isolated from root; S: bacteria isolated from shoot.

Discussion
Plant-associated endophytic bacteria are vital to the health of plants.They have been thought to be a valuable source of physiologically active chemicals because they create a variety of beneficial metabolites [19,27,49].Furthermore, some genetic explanations for the endophytic lifestyle of this bacterium have been offered by the whole-genome gene content study of plant-associated bacteria.The gene content analysis identified genes involved in motility, biofilm production, siderophore biosynthesis, chemotaxis, and osmoprotectant production, indicating their potential benefit for plant performance [50,51].This research is the first analysis of endophytic bacteria found in fennel (Foeniculum vulgare Mill.) growing in the desert Ugam-Chatkal State Biosphere Reserve in Uzbekistan.Profiling of endophytic bacteria isolated from the roots and shoots of fennel demonstrated that these included 18 isolates belonging to the genera Bacillus (5), Pseudomonas (6), Brevibacterium (2), Enterobacter (2), Klebsiella (2), and Stenotrophomonas (1).Similar bacterial species were isolated from the tissues of other medicinal plants, e.g., Bacillus megaterium from Lonicera japonica [52], Enterobacter cloacae from Tridax procumbens Linn.[53], Bacillus aryabhattai from Pterocarpus santalinus [54], Brevibacterium frigoritolerans from Ferula songorica [55], or Stenotrophomonas maltophilia from Armoracia rusticana [20].Notably, we observed Bacillus megaterium, Bacillus aryabhattai, and [Brevibacterium] frigoritolerans both in the roots and the shoots of fennel, which can be the result of the chemotactic movement of bacteria toward plant roots in response to exudates released by the plant.[56].The number of diverse isolates from shoots was higher than from roots.However, the diversity of culturable bacteria in plants represents only a fraction of the total microbial diversity.Advanced techniques like metagenomics and high-throughput sequencing are essential to capture a more comprehensive picture of the microbial communities associated with plants.Our study focused on the plant-beneficial traits of culturable bacteria associated with plants.Shi et al. [57] studied the total microbial community in potato tissues using Illumina MiSeq sequencing and found a higher diversity of bacteria species in roots than in shoots.The higher microbial diversity in roots compared to shoots is a result of the nutrient-rich environment, direct soil contact, favorable microenvironmental conditions, symbiotic relationships, and constant exposure to a diverse soil microbiome.
Endophytes support plant health by enhancing nutrient acquisition, promoting growth, suppressing diseases, increasing abiotic stress tolerance, and providing disease control.They exhibit several traits that help plants thrive.In our study, several bacterial endophytes showed antagonistic action against the plant pathogenic fungi F. oxysporum, F. solani, and R. solani.The antagonistic activity of endophytes reduces pathogen load and contributes to overall plant health.We did not find any correlation between the source of bacteria isolation (roots or shoots) and their antifungal activity.There were four isolates from roots and four from shoots with antifungal activity against F. culmorum and F. solani, and four isolates from roots and six from shoots with antifungal activity against R. solani.The different number of isolates from roots and shoots with activity against R. solani is due to two active isolates (BRN1 and BRN3) being found both in roots and shoots.Higher percentages of endophytes with antifungal characteristics were observed in previous studies on Chelidonium majus L. [58] and Hypericum perforatum-associated bacteria [16,32].There is evidence that the physiological processes of endophytic bacteria residing inside plant tissue may be influenced by the biologically active components of medicinal plants [27,39,59].Mehanni and Safwat [59] argued that endophytic bacteria may exhibit comparable biological activity and metabolite production to those of their hosts.The claim was validated by the research conducted by Koberl et al. [27] concerning endophytic bacteria extracted from the medicinal herbs Solanum distichum, Matricaria chamomilla, and Calendula officinalis, as well as endophytic bacteria isolated from Hypericum perforatum, which exhibited antifungal properties as their host.Furthermore, research revealed that fungal pathogens might be effectively suppressed without seriously harming the host by utilizing antagonistic characteristics of endophytic bacteria [60][61][62].Endophytes associated with Monarda citriodora, for instance, demonstrated antagonistic action against Fusarium oxysporum, while F. redolens demonstrated potential for biocontrol [28].
The antagonism of endophytes against plant pathogens is mediated through several well-defined mechanisms such as the synthesis of siderophores, enzymes that break down fungal cell walls, and hydrogen cyanide (HCN) [63][64][65].Chitinase, protease, glucanase, and lipase are the four tested enzymes that break down fungal cell walls.For example, chitinase can break down the essential component of the fungal cell wall, protease can break down fungal proteins, lipase can break down some lipids associated with the fungal cell wall, and β-1,3-glucanase can break down cell wall carbohydrates [16].Of the fifteen bacterial strains, eleven produced at least one of these enzymes.Our findings on the antagonistic activity of endophytes against plant pathogens are well-supported by various studies.In our previous study, the bacterial strains S. plymuthica RR2-5-10 and P. extremorientalis TSAU20 were able to produce the cell wall-degrading enzyme protease and showed biological control of cucumber root rot caused by Fusarium solani [60].Nineteen of the bacterial strains showed evidence of producing hydrogen cyanide (HCN), a process that also inhibits soil-borne pathogens [66].According to Michelsen and Stougaar [67], isolates of Pseudomonas fluorescens that produced hydrogen cyanide (HCN) impeded Rhizoctonia solani and Pythium aphanidermatum's hyphal development.
It is known that beneficial bacteria can produce phytohormones such as auxins (e.g., indole-3acetic acid), gibberellins, and cytokinins, which promote plant growth and development.Eight of the fifteen bacterial strains we studied produced IAA and induced the growth of the fennel seedlings' roots or shoots.Several studies have documented the synthesis of indole-3-acetic acid (IAA) by endophytic bacteria linked to different medicinal plants, including Thymus vulgaris, Majorana hortensis, Ocimum basilicum, Melissa officinalis, Marrubium vulgare, Solidago virgaurea, Melilotus officinalis, and Matricaria chamomilla [68].In pot trials, the endophytic bacteria isolated from Cassia occidentalis promoted mung bean plant growth by producing IAA [69].Phytohormones play crucial roles in regulating plant growth and development processes such as cell elongation, division, and differentiation.The modest increases in fennel growth parameters observed in this study could be attributed to the endogenous production of such hormones by the endophytic bacteria, which might have influenced root and shoot development [70][71][72][73].
Ethylene regulates plant responses to abiotic stresses such as high salinity, extreme temperatures, and heavy metals.The enzyme ACC deaminase is produced by plant-associated bacteria and has the ability to reduce levels of the ethylene precursor, ACC (1-aminocyclopropane-1-carboxylic acid), within plant tissues.By lowering ACC levels, ACC deaminase effectively decreases ethylene production in plants [74].Seven of the fifteen endophytic bacteria studied were capable of producing ACC deaminase.By reducing ethylene levels, ACC deaminase can help plants better tolerate these stresses.Although this study did not specifically measure stress parameters, the presence of endophytes might have contributed to a more robust stress response, allowing fennel plants to allocate resources more efficiently toward growth.In our previous study, the ACC deaminase-producing bacterial strains P. putida TSAU1 and P. aureantiaca TSAU22 stimulated the wheat root system in saline soils [47].
Eight out of fifteen bacterial strains produced siderophores.Microbial siderophores play an important role as determinants of biocontrol activity and influence the iron nutrition of plants [75,76].Seven out of fifteen bacterial strains possessed phosphate-solubilizing activity.Phosphate-solubilizing bacteria improve plants' phosphate nutrition by solubilizing insoluble phosphates in the soil and increasing the amount of phosphorus available for plants [77].
These traits, often exhibited by beneficial bacteria, can improve the nutrient availability in the rhizosphere, thereby promoting better growth and development of the plant.In the case of fennel, the observed increase in shoot and root length and dry weight suggests a potential improvement in nutrient uptake efficiency facilitated by the introduced endophytes.In this study, the introduction of endophytic bacteria into fennel seeds demonstrated positive, albeit modest, effects on the growth parameters of the plant.
Numerous papers have documented how endophyte inoculation improves plant growth.For example, Sudarshna and Sharma [78] reported that endophytic bacteria isolated from the medicinal plant Trillium govanianum increased plant growth and nutrient uptake of the plant under field conditions.The bacterial isolates demonstrated P solubilization activity and production of IAA, siderophore, and ACC deaminase.Similar results were obtained by Deepa et al. [79], whereas bacterial endophytes from Pelargonium graveolens demonstrated plant-beneficial traits and increased plant biomass and content of the essential oils geraniol and citronellol.

Conclusions
For the first time, endophytic bacteria from fennel (Foeniculum vulgare Mill.) samples taken from Uzbekistan's Ugam-Chatkal State Biosphere Reserve have been isolated, identified, and characterized in this work.Species belonging to Bacillus, Pseudomonas, Brevibacterium, Enterobacter, Klebsiella, and Stenotrophomonas were isolated and identified.In addition to demonstrating antifungal action against plant pathogenic fungi, the bacterial strains associated with fennel were found to be capable of synthesizing chitinase, protease, glucanase, lipase, HCN, siderophores, IAA, and ACC deaminase.According to our research, antimicrobial-rich medicinal plants may serve as a reservoir for microorganisms that exhibit antagonistic action against plant fungal pathogens, making them attractive options for the management of fungal diseases.They can also serve as an active part of biopreparation improving plant growth.These results also indicate that more investigation is required to determine how endophytic bacteria with particular plant growth promoting properties affect plant development and fungal disease control in field and pot studies.Further research should aim to optimize the use of endophytes to maximize their benefits and better understand their interactions with medicinal plants.

Table 1 .
Sequence similarities of endophyte bacteria isolated from the root system of fennel (Foeniculum vulgare Mill.) with sequences registered in GenBank.

Table 2 .
Sequence similarities of endophyte bacteria isolated from shoots of fennel (Foeniculum vulgare Mill.) with sequences registered in GenBank.
Figure 2. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences isolated from endophytic bacteria of fennel (Foeniculum vulgare Mill.), showing the relationship of isolated strains to their closest relatives in GenBank.

Table 4 .
Length and dry weight of shoot and root of fennel (Foeniculum vulgare Mill.) when seeds were inoculated with endophytic bacteria.Plants were grown in pots for two weeks.